Part:BBa_K523014

Plac + LacZ + bglX

The E. coli periplasmic β-glucosidase gene bglX under the control of the lac promoter. The native ribosome binding site is present.

Usage and Biology

The BglX β-glucosidase is expected to be localised to the periplasm. It ought to be capable of breaking down cellobiose.

Experiment

We (Edinburgh 2011) conducted two assays, comparing the activity of this part (Plac-bglX) with that of the exoglucanase cex under the control of the lac promoter (BBa_K523016) on two different substrates:

• 4-methylumbelliferyl β- D- glucuronide (MUG, left photo). This substrate is a cellobiose analog.
• 4-methylumbelliferyl β- D- cellobioside (MUC, right photo). This substrate is larger and is more like a cellulose analog.

Both substrates produce a fluorescent product when cleaved. Our plates below show the results of placing cell lysate and cell debris on an MUG plate and an MUC plate. Present on both plates are:

• Left side of plate: JM109 expressing this part, K523014
• Right side of plate: JM109 expressing exoglucanase cex, BBa_K523016
• Bottom of plate: JM109 cells

MUG assay. bglX on left, cex on right.		MUC assay. bglX on left, cex on right.

As can be seen, bglX is capable of degrading MUG (the cellobiose analog) while exoglucanase displays much weaker activity. By contrast, exoglucanase is much better at degrading MUC.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 607
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2266
  Illegal AgeI site found at 2488
  Illegal AgeI site found at 2677
  
• 1000
  COMPATIBLE WITH RFC[1000]